Purified thermostable Pyrococcus furiosus DNA polymerase I

ABSTRACT

Purified thermostable  Pyrococcus furiosus  DNA polymerase that migrates on a non-denaturing polyacrylamide gel faster than phosphorylase B and Taq polymerase and more slowly than bovine serum albumin and has an estimated molecular weight of 90,000–93,000 daltons when compared with a Taq polymerase standard assigned a molecular weight of 94,000 daltons.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/244,889, filed Feb. 5, 1999, now U.S. Pat. No. 6,489,150, which is acontinuation of application U.S. application Ser. No. 07/803,627, filedDec. 2, 1991, now U.S. Pat. No. 5,948,663, which is acontinuation-in-part of U.S. application Ser. No. 07/776,552, filed Oct.15, 1991, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 07/657,073, filed Feb. 19, 1991 now abandoned,which is continuation-in-part of U.S. application Ser. No. 07/620,568,filed Dec. 3, 1990, now abandoned. U.S. application Ser. No. 09/244,889is being relied upon and is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a thermostable enzyme having DNApolymerase I activity useful in nucleic acid synthesis by primerextension reaction.

Background

The archaebacteria are a recently discovered group of microorganismsthat grow optimally at temperatures above 80° C. Some 20 species ofthese extremely thermophilic bacteria-like organisms have been isolated,mainly from shallow submarine and deep sea geothermal environments. Mostof the archaebacteria are strict anaerobes and depend on the reductionof elemental sulfur for growth.

The archaebacteria include a group of “hyperthermophiles” that growoptimally around 100° C. These are presently represented by threedistinct genera, Pyrodictium, Pyrococcus, and Pyrobaculum. Phyodictiumbrockii (T_(opt) 105° C.) is an obligate autotroph which obtains energybe reducing S^(O) to H₂S with H₂, while Pyrobaculum islandicum (T_(opt)100° C.) is a faculative heterotroph that uses either organic substratesor H₂ to reduce S^(O). In contrast, Pyrococcus furiosus (T_(opt) 100°C.) grows by a fermentative-type metabolism rather than by S^(O)respiration. It is a strict heterotroph that utilizes both simple andcomplex carbohydrates where only H₂ and CO₂ are the detectable products.The organism reduces elemental sulfur to H₂S apparently as a form ofdetoxification since H₂ inhibits growth.

The discovery of microorganisms growing optimally around 100° C. hasgenerated considerable interest in both academic and industrialcommunities. Both the organisms and their enzymes have the potential tobridge the gap between biochemical catalysis and many industrialchemical conversions. However, knowledge of the metabolism of thehyperthermophilic microorganisms is presently very limited.

The polymerase chain reaction (PCR) is a powerful method for the rapidand exponential amplification of target nucleic acid sequences. PCR hasfacilitated the development of gene characterization and molecularcloning technologies including the direct sequencing of PCR amplifiedDNA, the determination of allelic variation, and the detection ofinfectious and genetic disease disorders. PCR is performed by repeatedcycles of heat denaturation of a DNA template containing the targetsequence, annealing of opposing primers to the complementary DNAstrands, and extension of the annealed primers with a DNA polymerase.Multiple PCR cycles result in the exponential amplification of thenucleotide sequence delineated by the flanking amplification primers.

An important modification of the original PCR technique was thesubstitution of Thermus aquaticus (Taq) DNA polymerase in place of theKlenow fragment of E. coli DNA pol I (Saiki, et al. Science,230:1350–1354 (1988)). The incorporation of a thermostable DNApolymerase into the PCR protocol obviates the need for repeated enzymeadditions and permits elevated annealing and primer extensiontemperatures which enhance the specificity of primer:templateassociations. Taq polymerase thus serves to increase the specificity andsimplicity of PCR.

Although Taq polymerase is used in the vast majority of PCR performedtoday, it has a fundamental drawback: purified Taq DNA polymerase enzymeis devoid of 3′ to 5′ exonuclease activity and thus cannot excisemisinserted nucleotides (Tindall, et al., Biochemistry, 29:5226–5231(1990)). Several independent studies suggest that 3′ to 5′exonuclease-dependent proofreading enhances the fidelity of DNAsynthesis. Reyland et al, J. Biol. Chem., 263:6518–6524, 1988; Kunkel etal, J. Biol. Chem., 261:13610–13616, 1986; Bernad et al, Cell,58:219–228, 1989. Consistent with these findings, the observed errorrate (mutations per nucleotide per cycle) of Taq polymerase isrelatively high; estimates range from 2×10⁻⁴ during PCR (Saiki et al.,Science, 239:487–491 (1988); Keohavaong et al. Proc. Natl. Acad. Sci.USA, 86:9253–9257 (1989)) to 2×10⁻⁵ for base substitution errorsproduced during a single round of DNA synthesis of the lacZ gene (Eckertet al., Nucl. Acids Res., 18:3739–3744 (1990)).

Polymerase induced mutations incurred during PCR increase arithmeticallyas a function of cycle number. For example, if an average of twomutations occur during one cycle of amplification, 20 mutations willoccur after 10 cycles and 40 will occur after 20 cycles. Each mutant andwild type template DNA molecule will be amplified exponentially duringPCR and thus a large percentage of the resulting amplification productswill contain mutations. Mutations introduced by Tag polymerase duringDNA amplification have hindered PCR applications which require highfidelity DNA synthesis.

SUMMARY OF THE INVENTION

A thermostable DNA polymerase from the hyperthermophilic, marinearchaebacterium, Pyrococcus furiosus (Pfu) has been discovered. Themonomeric, multifunctional enzyme possesses both DNA polymerase and 3′to 5′ exonuclease activities. The polymerase is extremely thermostablewith a temperature optimum near 75° C. The purified enzyme functionseffectively in the polymerase chain reaction (PCR). In addition, resultsfrom PCR fidelity studies indicate that Pyrococcus furiosus DNApolymerase yields amplification products containing 12 fold lessmutations than reaction products from similar amplifications performedwith Taq DNA polymerase. The 3′ to 5′ exonuclease dependent proofreadingactivity of Pfu DNA polymerase will excise mismatched 3′ terminalnucleotides from primer:template complexes and correctly incorporatenucleotides complementary to the template strand.

Unlike Taq DNA polymerase, Pfu DNA polymerase does not possess 5′ to 3′exonuclease activity. Pfu, like Taq and Vent polymerases, does exhibit apolymerase dependent 5′ to 3′ strand displacement activity. Pfu DNApolymerase remains greater that 95% active after one hour incubation at95° C. In contrast, Vent polymerase [New England Biolabs (NEB) Beverly,Mass.] looses greater than 50% of its polymerase activity after one hourincubation at 95° C. Pfu DNA polymerase is thus unexpectedly superior toTaq and Vent DNA polymerases in amplification protocols requiring highfidelity DNA synthesis.

Thus, the present invention contemplates a purified thermostable P.furiosus DNA polymerase I (Pfu DNA Pol I or Pyro polymerase) having anamino terminal amino acid residue sequence represented by the formulashown in SEQ ID NO 1, having 775 amino acid residues.

The apparent molecular weight of the native protein is about90,000–93,000 daltons as determined by SDS-PAGE under non-denaturing(non-reducing) conditions using Taq polymerase as a standard having amolecular weight of 94,000 daltons. In preferred embodiments, the Pyropolymerase is isolated from P. furiosus, and more preferably has aspecific 3′ to 5′ exonuclease activity.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 illustrates the molecular weight determination of Pyro DNApolymerase compared to Taq DNA polymerase by SDS-PAGE analysis (8–16%gradient gel, under non-reducing conditions). High molecular weightstandards (Sigma Chem. Co., St. Louis, Mo.) are electrophoresed in lanes1 and 6. The molecular weights of Phosphorylase B (97,200 daltons) andbovine serum albumin (66,000 daltons) in lane 6 are indicated by arrows.Sequencing grade Taq DNA polymerase which has been modified (PromegaBiotech, Madison, Wis.) has an apparent molecular weight of 80,000daltons (lane 2). Taq DNA polymerases from Cetus (Emeryville, Calif.)and Stratagene (La Jolla, Calif.) are electrophoresed in lanes 3 and 4,respectively, each with an apparent molecular weight of 94,000 daltons.In lane 5, Pyro DNA polymerase exhibits a molecular weight of90,000–93,000 daltons.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

As used herein, “cell”, “cell line”, and “cell culture” can be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” includes the primarysubject cell and cultures derived therefrom without regard for thenumber of transfers. It is also understood that all progeny may not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same functionality as screenedfor in the originally transformed cell are included.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for procaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and the like. Eucaryotic cells are known to utilizepromoters, polyadenylation signals, and enhancers.

The term “expression system” refers to DNA sequences containing adesired coding sequence and control-sequences in operable linkage, sothat hosts transformed with these sequences are capable of producing theencoded proteins. In order to effect transformation, the expressionsystem may be included on a vector; however, the relevant DNA may thenalso be integrated into the host chromosome.

The term “gene” as used herein refers to a DNA sequence that encodes apolypeptide.

“Operably linked” refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence “operablylinked” to control sequences refers to a configuration wherein thecoding sequences can be expressed under the direction of the controlsequences.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides and/or ribonucleotides,preferably more than three. Its exact size will depend on many factors,which in turn depend on the ultimate function or use of theoligonucleotide. The oligonucleotide may be derived synthetically or bycloning.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally or produced synthetically, which is capable ofacting as a point of initiation of nucleic acid synthesis when placedunder conditions in which synthesis of a primer extension product whichis complementary to a nucleic acid strand is induced, i.e., in thepresence of four different nucleotide triphosphates and thermostableenzyme in an appropriate buffer (‘buffer” includes pH, ionic strength,cofactors, etc.) and at a suitable temperature. For Pyro polymerase, thebuffer herein preferably contains 1.5–2 mM of a magnesium salt,preferably MgCl₂, 150–200 μM of each nucleotide, and 1 uM of eachprimer, along with preferably 50 mM KCl, 20 mM Tris buffer, pH 8–8.4,and 100 μg/ml gelatin.

The primer is preferably single-stranded for maximum efficiency inamplification, but may alternatively be double-stranded. Ifdouble-stranded, the primer is first treated to separate its strandsbefore being used to prepare extension products. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of thethermostable enzyme. The exact lengths of the primers will depend onmany factors, including temperature, source of primer and use of themethod. For example, depending on the complexity of the target sequence,the oligonucleotide primer typically contains 15–25 nucleotides,although it may contain more or few nucleotides. Short primer moleculesgenerally require colder temperatures to form sufficiently stable hybridcomplexes with template.

The primers herein are selected to be “substantially” complementary tothe different strands of each specific sequence to be amplified. Thismeans that the primers must be sufficiently complementary to hybridizewith their respective strands. Therefore, the primer sequence need notreflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thestrand to be amplified to hybridize therewith and thereby form atemplate for synthesis of the extension product of the other primer.However, for detection purposes, particularly using labeledsequence-specific probes, the primers typically have exactcomplementarity to obtain the best results.

As used herein, the term “thermostable enzyme” refers to an enzyme whichis stable to heat and is heat resistant and catalyzes (facilitates)combination of the nucleotides in the proper manner to form the primerextension products that are complementary to each nucleic acid strand.Generally, the synthesis will be initiated at the 3′ end of each primerand will proceed in the 5′ direction along the template strand, untilsynthesis terminates, producing molecules of different lengths.

The thermostable enzyme herein must satisfy a single criterion to beeffective for the amplication reaction, i.e., the enzyme must not becomeirreversibly denatured (inactivated) when subjected to the elevatedtemperatures for the time necessary to effect denaturation ofdouble-stranded nucleic acids. Irreversible denaturation for purposesherein refers to permanent and complete loss of enzymatic activity. Theheating conditions necessary for denaturation will depend, e.g., on thebuffer salt concentration and the length and nucleotide composition ofthe nucleic acids being denatured, but typically range from about 90 toabout 96° C. for a time depending mainly on the temperature and thenucleic acid length, typically about 0.5 to four minutes. Highertemperatures may be tolerated as the buffer salt concentration and/or GCcomposition of the nucleic acid is increased. Preferably, the enzymewill not become irreversibly denatured at about 90–100° C.

The thermostable enzyme herein preferably has an optimum temperature atwhich it functions that is higher than about 40° C., which is thetemperature below which hybridization of primer to template is promoted,although, depending on (1) magnesium and salt, concentrations and (2)composition and length of primer, hybridization can occur at highertemperature (e.g., 45–70° C.). The higher the temperature optimum forthe enzyme, the greater the specificity and/or selectivity of theprimer-directed extension process. However, enzymes that are activebelow 40° C., e.g., at 37° C., are also with the scope of this inventionprovided they are heat-stable. Preferably, the optimum temperatureranges from about 50° to 90° C., more preferably 60–80° C.

Amino Acid Residue: The amino acid residues described herein arepreferred to be in the “L” isomeric form. However, residues in the “D”isomeric form can be substituted for any L-amino acid residue, as longas the desired functional property is retained by the polypeptide. NH2refers to the free amino group present at the amino- or carboxy-terminusof a polypeptide. COOH refers to the free carboxy group present at thecarboxy terminus of a polypeptide. The amino-terminal NH₂ group andcarboxy-terminal COOH group of free polypeptides are typically not setforth in a formula. A hyphen at the amino- or carboxy-terminus of asequence indicates the presence of a further sequence of amino acidresidues or a respective NH₂ or COOH terminal group. In keeping withstandard polypeptide nomenclature, J. Biol. Chem., 243:3552–59 (1969)and adopted at 37 CFR §1.822(b)(2), abbreviations for amino acidresidues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteineIt should be noted that all amino acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.

Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1′ carbon of thepentose) and that combination of base and sugar is a nucleoside. Whenthe nucleoside contains a phosphate group bonded to the 3′ or 5′position of the pentose it is referred to as a nucleotide. A sequence ofoperatively linked nucleotides is typically referred to herein as a“base sequence” or “nucleotide sequence”, and is represented herein by aformula whose left to right orientation is in the conventional directionof 5′-terminus to 3′-terminus.

Base Pair (bp): A partnership of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a double stranded DNA molecule.

B. Pfu DNA Polymerase I (Pyro Polymerase)

Pyro polymerase, the thermostable DNA polymerase of the presentinvention, can be obtained from any source and can be a native orrecombinant protein. A preferred Pyro polymerase is isolated fromPyrococcus furiosus. P. furiosus is available from Dentsche Sammlung VonMicroorganismen (DSM), Grise-Bach StraSSE 8, d-3400 Göttengen, FRG,under the accession number DSM-6217.

For isolating the native protein from P. furiosus cells, such cells aregrown using any suitable technique. A variety of such techniques havebeen reported, those preferred being described by Fiala et al., Arch.Microbiol, (1986) 145:56–61, and Bryant et al., J. Biol. Chem., (1989)264:5070–5079, the disclosures of which are incorporated herein byreference.

After cell growth, the isolation and purification of Pyro polymerasetakes place in about 3 stages, each of which is performed at, andpreferably below, room temperature, preferably about 4° C.

In the first step, the cells are concentrated from the growth medium,typically by centrifugation or filtration.

In the second step, the cells are lysed and the supernatant issegregated and recovered from the cellular debris. Lysis is typicallyaccomplished by mechanically applying sheer stress and/or enzymaticdigestion. Segregation of the supernatant is usually accomplished bycentrifugation.

The third step removes nucleic acids and some protein. The supernatantfrom the second step is applied to an agarose resin strong anionicexchange column, such as Q-sepharose from Pharmacia (Piscataway, N.J.)equilibriated with column buffer [50 mM tris-hydroxymethylaminomethane(Tris), pH 8.2, 10 mM beta mercaphroethanol, and 1 mMethylenediaminetetraacetic acid (EDTA)]. The supernatant is washedthrough the column with the column buffer and the pass-through andwashes are collected and centrifuged to remove any insoluble material.The supernatant is segregated, usually dialyzed, and then recovered toform a fraction containing partially purified Pyro polymerase.

The fourth step removes substantially all (90%) of the remainingcontaminating proteins and comprises applying the fraction recoveredfrom step three to a phosphocellulose column equilibriated with thebefore described column buffer. The column is washed with the columnbuffer until the optical density of the wash eluate is at the bufferbaseline at 280 nm. The immobilized Pyro polymerase is thereafter elutedwith a linear salt gradient comprising O M to about 0.7 M salt dissolvedin the column buffer, the salt being NaCl, KCl, and the like. Proteineluted from the column at about 200 mM salt typically contains thehighest concentrations of assayable Pyro polymerase.

In preferred embodiments, the Pyro polymerase preparation obtained fromthe fourth step is further purified in a fifth step by FPLCchromatography through a high performance cation exchange column, suchas the Mono S column available from Pharmacia, Piscataway, N.J.,equilbriated with the before described column buffer. After application,the column is washed to remove non-bound contaminants. The immobilizedPyro polymerase is then eluted with the before-described linear saltgradient at about 120 nM salt concentration. The Pyro polymerase eluateis then typically dialysed against the column buffer to remove excesssalt. A stabilizing agent, such as glycerol, can be added to thepreparation at this time to facilitate low temperature storage.Typically, the fraction is again dialyzed against a low salt buffer,e.g., 50 mM Tris pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1% Tween20, and 0.1% non-idet P40.

In further preferred embodiments the Pyro polymerase preparation of stepfive is applied to a crosslinked agarose affinity column, such as theAffi-Gel Blue column available from BioRad, Richmond, Calif.,equilibrated with the before-described column buffer. Non-bound proteinis washed from the column and the Pyro polymerase is eluted with thebefore-described salt gradient with the Pyro polymerase typically beingrecovered at about 280 mM salt concentration. Thereafter, the Pyropolymerase preparation is usually concentrated about 5–10 fold anddialysed against column buffer. Typically, a stabilizing agent, such asglycerol, is added to the preparation to facilitate low temperaturestorage.

The amino-terminal amino acid residue sequence of Pyro polymerase can bedetermined by any suitable method, such as by automated Edmandegradation, and the like. The amino acid residue sequence of apreferred Pyro polymerase is shown in SEQ ID NO 1 from residue 1 to 775.

The molecular weight of the dialyzed product may be determined by anytechnique, for example, by sodium dodecylsulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) using protein molecular weight markers.Native Pyro polymerase purified by the above method has a relativemolecular weight, determined by SDS-PAGE under non-reducing conditions,of about 90,000–93,000 daltons.

In preferred embodiments, Pyro polymerase is used in combination with athermostable buffering agent such as TAPS(N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid; ([2-Hydroxy-1,1-bis(hydroxy-methyl)-ethyl]amino-1-propanesulfonic acid), availablefrom Sigma, St. Louis, Mo. (Catalog P7905).

C. Recombinant Pyro Polymerase

Pyro polymerase can also be produced by recombinant DNA (rDNA)techniques, as the gene encoding the enzyme can be cloned from P.furiosus genomic DNA. Thus, the present invention also contemplates aDNA segment consisting essentially of a sequence of nucleotide basesequence encoding a Pyro polymerase of this invention. An exemplary DNAsequence, obtained from the native gene, coding for a preferred Pfu Iprotein is shown in SEQ ID NO 2 from nucleotide base 224 to base 2548,which spans the coding portion of SEQ ID NO 2.

The isolated gene can be operably linked to an expression system to forman rDNA capable of expressing, in a compatible host, Pyro polymerase.

Of course, modifications to the primary structure itself by deletion,addition, or alteration of the amino acids incorporated into the proteinsequence during translation can be made without destroying the activityof the protein. Such substitutions or other alterations result inproteins having an amino acid sequence encoded by DNA falling within thecontemplated scope of the present invention.

1. Cloning and Expression of the Pyro Polymerase Gene

Polyclonal antiserum from rabbits immunized with the purified90,000–93,000 dalton polymerase of this invention can be used to probe aP. furiosus partial genomic expression library to obtain the appropriatecoding sequence as described below. The cloned genomic sequence can beexpressed as a fusion protein, expressed directly using its own controlsequences, or expressed by constructions using control sequencesappropriate to the particular host used for expression of the enzyme.

Thus, the complete coding sequence for Pyro polymerase from whichexpression vectors applicable to a variety of host systems can beconstructed and the coding sequence expressed. It is also evident fromthe foregoing that portions of the Pyro polymerase-encoding sequence areuseful as probes to retrieve other similar thermostablepolymerase-encoding sequences in a variety of Archaebacteria species,particularly from other Pyrococcus species and P. furiosus strains.Accordingly, portions of the genomic DNA encoding at least sixcontiguous amino acids can be synthesized and used as probes to retrieveadditional DNAs encoding an Archaebacteria thermostable polymerase.Because there may not be a precisely exact match between the nucleotidesequence in the P. furiosus form described herein and that in thecorresponding portion of other species or strain, oligomers containingapproximately 18 nucleotides (encoding the six amino acid stretch) areprobably necessary to obtain hybridization under conditions ofsufficient stringency to eliminate false positives. The sequencesencoding six amino acids would supply information sufficient for suchprobes.

Exemplary degenerate Pfu DNA polymerase I gene probes are shown in Table1.

TABLE 1 Degenerate Pfu DNA Polymerase I Gene Probes¹        A T  T  T  I 1) 5′GACTACATCACCGAIGAIGG3′     T  T        A  A 2)5′CCCTCCTCIGTIATGTAGTC3′     T  A  A  T 3) 5′TTCAAGAAGAACGG-3′    A  T  T  A 4) 5′CCGTTCTTCTTGAA-3′ ¹Nucleotide sequence degeneratesubstitutions are bases directly above the positions in the sequencewhere the substitutions were made. In some cases, degeneracy wasaccomplished by substituting inosine (I) in the sequence.

A preferred and exemplary cloning protocol for isolation of a pfu pol Igene is described in the Examples. From the clone pF72 described in theExamples, the nucleotide sequence of a preferred gene encoding pfu pol Iwas described and is shown in SEQ ID NO 2, and can be utilized for theproduction of recombinant Pyro polymerase.

In general terms, the production of a recombinant form of Pyropolymerase typically involves the following:

First, a DNA is obtained that encodes the mature (used here to includeall muteins) enzyme or a fusion of the Pyro polymerase either to anadditional sequence that does not destroy its activity, or to anadditional sequence cleavable under controlled conditions (such astreatment with peptidase) to give an active protein. If the sequence isuninterrupted by introns it is suitable for expression in any host. Thissequence should be in an excisable and recoverable form.

The excised or recovered coding sequence is then preferably placed inoperable linkage with suitable control sequences in a replicableexpression vector. The vector is used to transform a suitable host andthe transformed host cultured under favorable conditions to effect theproduction of the recombinant Pyro polymerase. Optionally the Pyropolymerase is isolated from the medium or from the cells; recovery andpurification of the protein may not be necessary in some instances,where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The constructions forexpression vectors operable in a variety of hosts are made usingappropriate replicons and control sequences, as set forth below.Suitable restriction sites can, if not normally available, be added tothe ends of the coding sequence so as to provide an excisable gene toinsert into these vectors.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, procaryotic, yeast, insect or mammalian cells are presentlyuseful as hosts. Procaryotic hosts are in general the most efficient andconvenient for the production of recombinant proteins and therefore arepreferred for the expression of Pyro polymerase.

2. Control Sequences and Corresponding Hosts

Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example, Bacillus subtillis, various species ofPseudomonas, or other bacterial strains. In such procaryotic systems,plasmid vectors that contain replication sites and control sequencesderived from species compatible with the host are used. For example, E.coli is typically transformed using derivatives of pBR322, a plasmidderived from an E. coli species by Bolivar, et al., Gene, (1977) 2:95and Sutcliffe, Nuc. Acids Res., (1978) 5:2721–28. pBR322 contains genesfor ampicillin and tetracycline resistance, and thus provides additionalmarkers that can be either retained or destroyed in constructing thedesired vector. Commonly used procaryotic control sequences, which aredefined herein to include promoters for transcription initiation,optionally with an operator, along with ribosome binding site sequences,include such commonly used promoters as the B-lactamase (penicillinase)and lactose (lac) promoter systems (Chang, et al., Nature, (1977)198:1056), the tryptophan (trp) promoter system (Goeddel, et al.,Nucleic Acids Res., (1980) 8:4057) and the lambda-derived P_(L) promoter(Shimatake, et al., Nature, (1981) 292:128) and N-gene ribosome bindingsite, which has been made useful as a portable control cassette (as setforth in U.S. Pat. No. 4,711,845), which comprises a first DNA sequencethat is the P_(L) promoter operably linked to a stream of a third DNAsequence having at least one restriction site that permits cleavage withsix bp 3′ of the N_(RBS) sequence. Also useful is the phosphatase A(phoA) system described by Change, et al. in European Patent PublicationNo. 196,864. However, any available promoter system compatible withprocaryotes can be used. Typical bacterial plasmids are pUCS, pUC9,pBR322 and pBR329 available from Bio-Rad Laboratories, (Richmond,Calif.) and pPL and pkk233-2, available from Pharmacia (Piscataway,N.J.) or Clone Tech (Palo Alto, Calif.).

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used, although a number of other strains are commonlyavailable. While vectors employing the 2 micron origin of replicationare illustrated (Broach, J. R., Meth. Enz., (1983) 101:307), otherplasmid vectors suitable for yeast expression are known (see, forexample, Stinchcomb, et al., Nature, (1979) 282:39, Tschempe, et al.,Gene, (1980) 10:157, Clarke, L., et al., Meth. Enz (1983) 101:300),Brake et al., Proc. Natl. Acad. Sci, USA, (1984) 81:4642–4647, andHalewell et al., Biotechnology, (1987) 5:363–366. Control sequences foryeast vectors include promoters for the synthesis of glycolytic enzymes(Hess, et al., J. Adv. Enzyme Reg., (1968) 7:149; Holland, et al.,Biotechnology (1978) 17:4900).

Additional promoters known in the art include the promoter for3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem., (1980)255:2073) and those for other glycolytic enzymes, such asglyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other promoters that have theadditional advantage of transcription controlled by growth conditionsare the promoter regions for alcohol dehydrogenase 2, iscoytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, and enzymes responsible for maltose and galactoseutilization (Holland, supra).

It is also believed that terminator sequences are desirable at the 3′end of the coding sequences. Such terminators are found in the 3′untranslated region following the coding sequences in yeast-derivedgenes. Many of the vectors illustrated contain control sequences derivedfrom the enolase gene containing plasmid peno46 (Holland, M. M., et al.,J. Biol. Chem., (1981) 256:1385) or the LEU2 gene obtained from YEp13(Broach, J., et al., Gene, (1978) 8:21); however, any vector containinga yeast-compatible promoter, origin of replication, and other controlsequences is suitable.

It is also, of course, possible to express genes encoding polypeptidesin eucaryotic host cell cultures derived from multicellular organisms.See, for example, Tissue Culture, Academic Press, Cruz and Patterson,editors (1973). Useful host cell lines include murine myelomas N51, VEROand HeLA cells, and Chinese hamster ovary (CHO) cells available from theATCC as CCL61, and NIH/3T3 mouse cells available from the ATCC asCRL1658. Expression vectors for such cells ordinarily include promotersand control sequences compatible with mammalian cells such as, forexample, the commonly used early and late promoters from Simian Virus 40(SV 40) (Fiers, et al., Nature, (1978) 273:113), or other viralpromoters such as those derived from polyoma, Adenovirus 2, bovinepapilloma virus, or avian sarcoma viruses, or immunoglobulin promotersand heat shock promoters. A system for expressing DNA in mammaliansystems using the BPV as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. General aspects of mammalian cell host system transformationshave been described in U.S. Pat. No. 4,399,216. It now appears, also,that “enhancer” regions are important in optimizing expression; theseare, generally, sequences found upstream of the promoter region. Originsof replication may be obtained, if needed, from viral sources. However,integration into the chromosome is a common mechanism for DNAreplication in eucaryotes.

Plant cells are also now available as hosts, and control sequencescompatible with plant cells such as the nopaline synthase promoter andpolyadenylation signal sequences (Depicker, A., et al., J. Mol. Appl.Gen., (1982) 1:561) are available. See, also, U.S. Pat. No. 4,962,028,U.S. Pat. No. 4,956,282, U.S. Pat. No. 4,886,753 and U.S. Pat. No.4,801,540.

Recently, in addition, expression systems employing insect cellsutilizing the control systems provided by baculovirus vectors have beendescribed (Miller, D. W., et al., in Genetic Engineering (1986) Setlow,J. K. et al., eds., Plenum Publishing, Vol. 8, pp. 277–297). See, also,U.S. Pat. No. 4,745,051 and No. 4,879,236. These systems are alsosuccessful in producing Pyro polymerase.

A preferred DNA segment containing both the pfu pol I coding portion andcontrol sequences at the 5′ and 3′ termini of the coding portion isshown in SEQ ID NO 2 from nucleotide base 1 to base 3499.

3. Transformations

The recombinant DNA molecules of the present invention are introducedinto host cells, via a procedure commonly known as transformation ortransfection. Transformation of appropriate host cells with arecombinant DNA molecule of the present invention is accomplished bywell known methods that typically depend on the type of vector used.With regard to transformation of procaryotic host cells or other cellsthat contain substantial cell wall barriers, see, for example, Cohen etal., Proc. Natl. Acad. Sci. USA, 69:2110 (1972); and Maniatis et al.,Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). With regard to transformation ofvertebrate cells with retroviral vectors containing rDNA, see, forexample, Sorge et al., Mol. Cell. Biol., 4:1730–37 (1984); and Wigler etal., Proc. Natl. Acad. Sci. USA, 76:1373–76 (1979).

Infection with Agrobacterium tumefaciens (Shaw, C. H., et al., Gene,(1983) 23:315) is used for certain plant cells. For mammalian cellswithout cell walls, the calcium phosphate precipitation method of Grahamand van der Eb, Virology (1978) 52:546 is preferred. Transformationsinto yeast are carried out according to the method of Van Solingen, P.,et al., J. Bact. (1977) 130:946 and Hsiao, C. L., et al., Proc. Natl.Acad. Sci. (USA), (1979) 76:3829.

Successfully transformed cells, i.e., cells that contain a recombinantDNA (rDNA) molecule of the present invention, are usually monitored byan appropriate immunological, hybridization or functional assay. Forexample, cells resulting from the introduction of an rDNA of the presentinvention can be cloned to produce monoclonal colonies. Cells from thosecolonies can be harvested, lysed and their DNA content examined for thepresence of the rDNA using a method such as that described by Southern,J. Mol. Biol., 98:503 (1975) or Berent et al., Biotech., 3:208 (1985).

In addition to directly assaying for the presence of rDNA, successfultransformation can be confirmed by well known immunological methods whenthe rDNA is capable of directing the expression of Pyro polymerase. Forexample, cells successfully transformed with a subject rDNA containingan expression vector produce a polypeptide displaying a characteristicantigenicity. Samples of a culture containing cells suspected of beingtransformed are harvested and assayed for a subject polypeptide (Pyropolymerase) using antibodies specific for that polypeptide antigen, suchas those produced by an appropriate hybridoma.

A particularly convenient assay technique involves fusing the Pyropolymerase-encoding DNA to a Lac Z gene in a suitable plasmid, e.g. pLG.Since the plasmid lacks a promoter and Shine-Dalgarno sequence, noβ-galactosidase is synthesized. However, when a portable promoterfragment is properly positioned in front of the fused gene, high levelsof a fusion protein having β-galactosidase activity should be expressed.The plasmids are used to transform Lac-bacteria which are scored forβ-galactosidase activity on lactose indicator plates. Plasmids havingoptimally placed promoter fragments are thereby recognized. Theseplasmids can then be used to reconstitute the fusion protein gene whichis expressed at high levels.

Thus, in addition to the transformed host cells themselves, cultures ofthe cells are contemplated as within the present invention. The culturesinclude monoclonal (clonally homogeneous) cultures, or cultures derivedfrom a monoclonal culture, in a nutrient medium. Nutrient media usefulfor culturing transformed host cells are well known in the art and canbe obtained from several commercial sources. In embodiments wherein thehost cell is mammalian, a “serum-free” medium is preferably used.

The present method entails culturing a nutrient medium containing hostcells transformed with a recombinant DNA molecule of the presentinvention that is capable of expressing a gene encoding a subjectpolypeptide. The culture is maintained for a time period sufficient forthe transformed cells to express the subject polypeptide. The expressedpolypeptide is then recovered from the culture.

Once a gene has been expressed in high levels, a DNA fragment containingthe entire expression assembly, e.g., promoter, ribosome-binding site,and fusion protein gene) may be transferred to a plasmid that can attainvery high copy numbers. For instance, the temperature-inducible “runawayreplication” vector pKN402 may be used. Preferably, the plasmid selectedwill have additional cloning sites which allow one to score forinsertion of the gene assembly. See, Bittner et al. Gene, 15:31 (1981).Bacterial cultures transformed with the plasmids are grown for a fewhours to increase plasmid copy number, e.g., to more than 1000 copiesper cell. Induction may be performed in some cases by elevatedtemperature and in other cases by addition of an inactivating agent to arepressor. Potentially very large increases in cloned fusion proteinscan be obtained in this way.

4. Construction of a Lambda Expression Library

The strategy for isolating DNA encoding desired proteins such as thePyro polymerase encoding DNA, using the bacteriophage vector lambdagt11, is as follows. A library can be constructed of EcoRI-flanked AluIfragments, generated by complete digestion of P. furiosus DNA, insertedat the EcoRI site in the lambda gt11 phage (Young and Davis, Proc. Natl.Acad. Sci. (USA), (1983) 80:1194–1198). Because the unique EcoRI site inthis bacteriophage is located in the carboxyl-terminus of theB-galactosidase gene, inserted DNA (in the appropriate frame andorientation) is expressed as protein fused with B-galactosidase underthe control of the lactose operon prompter/operator.

Genomic expression libraries are then screened using the antibody plaquehybridization procedure. A modification of this procedure, referred toas “epitope selection,” uses antiserum against the fusion proteinsequence encoded by the phage, to confirm the identification ofhybridized plaques. Thus, this library of recombinant phages could bescreened with antibodies that recognize the 90,000–93,000 dalton Pyropolymerase in order to identify phage that carry DNA segments encodingthe antigenic determinants of the Pyro polymerase protein.

Approximately 2×10⁵ recombinant phage are screened using rabbit Pyropolymerase antiserum. In this primary screen, positive signals aredetected and one or more of these plaques are purified from candidateplaques which failed to react with preimmune serum and reacted withimmune serum and analyzed in some detail. Anti-Pyro polymeraseantibodies can be prepared by a number of known methods, see, forexample, U.S. Pat. No. 4,082,735, U.S. Pat. No. 4,082,736, and U.S. Pat.No. 4,493,795.

To examine fusion proteins produced by the recombinant phage, lysogensof the phage in the host Y1089 are produced. Upon induction of thelysogens and gel electrophoresis of the resulting proteins, each lysogenmay be observed to produce a new protein, not found in other lysogens,or duplicate sequences may result. Phage containing positive signals arepicked. Typically, one positive plaque is picked for furtheridentification and replated at lower densities to purify recombinantsand the purified clones are analyzed by size class via digestion withEcoRI restriction enzyme. Probes can then be made of the isolated DNAinsert sequences and labeled appropriately and these probes can be usedin conventional colony or plaque hybridization assays described inManiatis et al., Molecular Cloning: A Laboratory Manual, (1982), thedisclosure of which is incorporated herein by reference. Particularhybridization conditions that may be used include incubating DNA at 42°C. in a hybridization buffer comprising 0.9 M NaCl and 50% formamide.Wash conditions that may be used after hybridization include incubatingDNA at 68° C. in a wash buffer comprising 0.015 M NaCl and 0.5% sodiumdodecyl sulfate.

5. Recombinant DNA Molecules

The present invention further contemplates a recombinant DNA (rDNA) thatincludes a Pyro polymerase-encoding DNA segment of the present inventionoperatively linked to a vector for replication and/or expression.Preferred rDNA molecules contain less than 50,000 nucleotide base pairs,usually less than 20,000 base pairs and preferably less than about10,000 base pairs. Preferably, a Pyro polymerase-encoding DNA of thisinvention is in the form of a plasmid, cosmid or phage.

A preferred rDNA molecule includes a nucleotide sequence shown in SEQ IDNO 2 from nucleotide base 224 to base 2548.

A rDNA molecule of the present invention can be produced by operativelylinking a vector to a DNA segment of the present invention.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked Preferred vectorsare those capable of autonomous replication and/or expression of nucleicacids to which they are operatively linked are referred to herein as“expression vectors”. As used herein, the term “operatively linked”, inreference to DNA segments, describes that the nucleotide sequence isjoined to the vector so that the sequence is under the transcriptionaland translation control of the expression vector and can be expressed ina suitable host cell.

As is well known in the art, the choice of vector to which a proteinencoding DNA segment of the present invention is operatively linkeddepends upon the functional properties desired, e.g., proteinexpression, and upon the host cell to be transformed. These limitationsare inherent in the art of constructing recombinant DNA molecules.However, a vector contemplated by the present invention is at leastcapable of directing the replication, and preferably also expression, ofa gene operatively linked to the vector.

In preferred embodiments, a vector contemplated by the present inventionincludes a procaryotic replicon, i.e., a DNA sequence having the abilityto direct autonomous replication and maintenance of the recombinant DNAmolecule extrachromosomally in a procaryotic host cell, such as abacterial host cell, transformed therewith. Such replicons are wellknown in the art. In addition, those embodiments that include aprocaryotic replicon may also include a gene whose expression confers aselective advantage such as amino acid nutrient dependency or drugresistance to a bacterial host transformed therewith as is well known,in order to allow selection of transformed clones. Typical bacterialdrug resistance genes are those that confer resistance to ampicillin,tetracycline, or kanamycin.

Those vectors that include a procaryotic replicon may also include aprocaryotic promoter capable of directing the expression (transcriptionand translation) of the gene transformed therewith. A promoter is anexpression control element formed by a DNA sequence that permits bindingof RNA polymerase and transcription to occur. Promoter sequencescompatible with bacterial hosts are typically provided in plasmidvectors containing convenient restriction sites for insertion of a DNAsegment of the present invention. Bacterial expression systems, andchoice and use of vectors in those systems is described in detail in“Gene Expression Technology”, Meth. Enzymol., Vol 185, Goeddel, Ed.,Academic Press, NY (1990).

Expression vectors compatible with eucaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form therecombinant DNA molecules of the present invention. Eucaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desiredgene. Typical of such vectors are pSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), and pTDT1 (ATCC,#31255).

In preferred embodiments, the eucaryotic cell expression vectors used toconstruct the recombinant DNA molecules of the present invention includea selectable phenotypic marker that is effective in a eucaryotic cell,such as a drug resistance selection marker or selective marker based onnutrient dependency. A preferred drug resistance marker is the genewhose expression results in neomycin resistance, i.e., the neomycinphosphotransferase (neo) gene. Southern et al., J. Mol. Appl. Genet.,1:327–341 (1982).

The use of retroviral expression vectors to form the rDNAs of thepresent invention is also contemplated. As used herein, the term“retroviral expression vector” refers to a DNA molecule that includes apromoter sequence derived from the long terminal repeat (LTR) region ofa retrovirus genome.

In addition to using strong promoter sequences to generate largequantities of mRNA coding for the expressed fusion proteins of thepresent invention, it is desirable to provide ribosome-binding sites inthe mRNA to ensure efficient translation. The ribosome-binding site inE. coli includes an initiation codon (AUG) and a sequence 3–9nucleotides long located 3–11 nucleotides upstream from the initiationcodon (the Shine-Dalgarno sequence). See, Shine et al., Nature, 254:34(1975). Methods for including a ribosome-binding site in mRNAscorresponding to the expressed proteins are described by Maniatis, etal. Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY pp. 412–417 (1982). Ribosome binding sites can bemodified to produce optimum configuration relative to the structuralgene for maximal expression of the structural gene. Halewell et al.,Nucl. Acid. Res., (1985) 13:2017–2034.

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniquesthat are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site-specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions that are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. Seee.g., New England Biolabs, Product Catalog. In general, completedigestion is obtained by admixing about 1 ug of plasmid or DNA sequencewith one unit of enzyme in about 20 ml of buffer solution. Incubationtimes of about one hour to two hours at about 37° C. are workable,although variations can be tolerated. After each incubation, protein isremoved by extraction with phenol/chloroform, and may be followed byether extraction, and the nucleic acid recovered from aqueous fractionsby precipitation with ethanol. If desired, size separations is found inMethods in Enzymology (1980) 65:499–560.

Restriction-cleaved fragments may be blunt-ended by treating with largefragment E. coli DNA polymerase I (Klenow) in the presence of the fourdeoxynucleotide triphosphates (dNTPs) using incubation times of about 15to 25 minutes at 20 to 25° C. in 50 mM Tris pH 7.6, 50 mM NaCl, 10 mMMgCl₂, 10 mM DTT and 50–100 μM dNTPs. The Klenow fragment fills in at 5′sticky ends, but chews back protruding 3′ single strands even though thefour dNTPs are present. If desired, selective repair can be performed bysupplying only one of the, or selected, dNTPs within the limitationsdictated by the nature of the sticky ends. After treatment with Klenow,the mixture is extracted with phenol/chloroform and ethanolprecipitated. Treatment under appropriate conditions with S1 nucleaseresults in hydrolysis of any single-stranded portion.

Synthetic oligonucleotides may be prepared using the triester method ofMatteucci, et al., (J. Am. Chem. Soc., (1981) 103:3185–3191) or usingautomated synthesis methods. Kinasing of single strands prior toannealing or for labeling is achieved using an excess, e.g.,approximately 10 units of polynucleotide kinase to 1 nM substrate in thepresence of 50 mM Tris, pH 7.6, 10 mN MgCl₂, 5 mM dithiothreitol, 1–2 mMATP. If kinasing is for labeling of probe, the ATP will contain highspecific activity ³²P.

Ligations are performed in 15–30 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01–0.02(Weiss) units TA DNA ligase at 0° C. (for “sticky end” ligation) or 1 mMATP, 0.3–0.6 (Weiss) units T4 DNA ligase at 14° C. (for “blunt end”ligation). Intermolecular “sticky end” ligations are usually performedat 33–100 μg/ml total DNA concentrations (5–100 nM total endconcentration). Intermolecular blunt end ligations (usually employing a10–30 fold molar excess of linkers) are performed at 1 μM total endsconcentration.

In vector construction employing “vector fragments”, the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) in orderto remove the 5′ phosphate and prevent religation of the vector. BAPdigestions are conducted at PH 8 in approximately 150 mM Tris, in thepresence of Na⁺ and Mg⁺² using about 1 unit of BAP per mg of vector at60° C. for about one hour. In order to recover the nucleic acidfragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors that have been double digested by additional restriction enzymedigestion of the unwanted fragments.

For portions of vectors derived from cDNA or genomic DNA that requiresequence modifications, sit-specific primer-directed mutagenesis isused. this technique is now standard in the art, and is conducted usinga primer synthetic oligonucleotide complementary to a single-strandedphage DNA to be mutagenized except for limited mismatching, representingthe desired mutation. Briefly, the synthetic oligonucleotide is used asa primer to direct synthesis of a strand complementary to the phage, andthe resulting double-stranded DNA is transformed into a phage-supportinghost bacterium. Cultures of the transformed bacteria are plated in topagar, permitting plaque formation from single cells that harbor thephage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.The plaques are transferred to nitrocellulose filters and the “lifts”hybridized with kinased synthetic primer at a temperature that permitshybridization of an exact match, but at which the mismatches with theoriginal strand are sufficient to prevent hybridization. Plaques thathybridize with the probe are then picked and cultured, and the DNA isrecovered.

6. Verification of Construction

Correct ligations for plasmid construction are confirmed by firsttransforming E. coli strain MM294, or other suitable host, with theligation mixture. Successful transformants are selected by ampicillin,tetracycline or other antibiotic resistance or using other markers,depending on the mode of plasmid construction, as is understood in theart. Plasmids from the transformants are then prepared according to themethod of Clewell, D. B., et al., Proc. Natl. Acad. Sci. (USA), (1969)62:1159, optionally following chloramphenicol amplication (Clewell, D.B., J. Bacteriol., (1972), 110:667). The isolated DNA is analyzed byrestriction digest mapping and/or sequenced by the dideoxy method ofSanger, F., et al., Proc. Natl. Acad. Sci. (USA), (1977) 74:5463 asfurther described by Messing, et al., Nucleic Acids Res., (1981) 9:309,or by the method of Maxam, et al., Methods in Enzymology, (1980) 65:499.

Host strains useful in cloning and expression are as follows:

For cloning and sequencing, and for expression of constructions undercontrol of most bacterial promoters, E. coli strain MM294 obtained fromE. coli Genetic Stock Center GCSC #6135, is particularly useful. Forexpression under control of the P_(L)N_(RBS) promoter, E. coli strainK12 MC1000 lambda lysogen, N₇N_(53c)I857 SusP₈₀ (ATCC 39531), may beused. Also useful is E. coli DG116, (ATCC 53606).

For M13 phage recombinants, E. coli strains susceptible to phageinfection, such as E. coli K12 strain DG98, are employed. The DG98strain has been deposited with ATCC Jul. 13, 1984 and has accessionnumber 39768.

The thermostable enzyme of this invention may be used for any purpose inwhich such enzyme is necessary or desirable. In a particularly preferredembodiment, the enzyme herein is employed in the amplification protocolset forth below.

EXAMPLES

The following examples are intended to illustrate, but not limit, thepresent invention.

1. Culturing of Pyrococcus furiosus and Preparation of Pf Cell Paste

The following describes how the hyperthermophilic archaebacterium, P.furiosus, is routinely grown in a 500 liter fermentor for the purpose ofobtaining cell mass in sufficient quantities for large scale proteinpurification. It is a modified version (Bryant et al., J. Biol. Chem.,264:5070–5079 (1989)) of the original protocol of Fiala et al., Arch.Microbiol., 145:56–61 (1986).

For culture maintenance, P. furiosus (DSM 3638) is routinely grown at85–88° C. as a closed static culture in 100 ml of the medium describedin Table 2.

TABLE 2 Maltose 5 g/l NH₄Cl 1.25 g/l Elemental Sulfur 5 g/l Na₂S 0.5 g/lSynthetic Sea Water¹ Vitamin mixture² 1 ml/l FeCl₃ 25 μM Na₂WO₄ 10 μMYeast Extract 0.01% ¹Synthetic Sea Water: NaCl, 13.8 g/l MgSO₄, 3.5 g/lMgCl₂, 2.7 g/l KCl, 0.3 g/l CaCl₂, 0.75 g/l KH₂PO₄, 0.5 g/l NaBr, 0.0–5g/l KI, 0.05 g/l H₃BO₃, 0.015 g/l Sodium citrate, 0.005 g/l ²Vitaminmixture [Balch et al., Microbiol. Rev., 43:260–296 (1979)]: Biotin, 2mg/l Folic acid, 2 mg/l Pyridoxine hydrochloride, 10 mg/l Thiaminehydrochloride, 5 mg/l Riboflavin, 5 mg/l Nicotinic acid, 5 mg/lDL-Calcium pantothenate, 5 mg/l Vitamin B₁₂, 0.1 mg/l p-Aminobenzoicacid, 5 mg/l Lipoic acid, 5 mg/l

Growth is monitored by the increase in turbidity at 600 nm. Cells can bestored in the same medium at 4° C. and remain viable for at least ayear, although periodic transfer is recommended.

Large scale (preparative) growth of P. furiosus was performed asfollows:

Growth medium according to Table 1, was prepared, except that thesulfide was replaced with titanium (III) nitrilotriacetate (finalconcentration, 30 μM as described in Moench et al., J. Microbiol. Meth.,1:199–202 (1983)] and the elemental sulfur is omitted. The medium wasthen sparged with Argon (Ar).

A two liter flask was inoculated with two 100 ml cultures. The two literculture was used as an inoculum for a 20 liter culture. Two 20 litercultures were used to inoculate a 500 liter culture. The culture wasmaintained at 88° C., bubbled with Ar (7.5 liters/min) and stirred atabout 50 rpm. After about 20 hours (A₆₀₀˜0.5) the cells were harvestedwith a Sharples continuous flow centrifuge at 100 liters/hour. The cellswere frozen in liquid N2 immediately after harvesting. The yield ofcells is typically 400–600 g wet weight.

It should be noted that P. furiosus has a fermentative type ofmetabolism and produces organic acids, CO₂ and H₂ as final products. H₂production inhibits growth, so cultures have to be sparged with Ar (orany inert gas) to remove H₂. Alternatively, elemental sulfur may beadded. In this case, the reductant that would otherwise be used togenerate H₂ is used to reduce elemental sulfur to H₂S. The addition ofelemental sulfur is convenient for small scale cultures in glassvessels, but its reduction cannot be used to remove inhibitory H₂ in 500liter stainless steel fermentors because of the corrosive nature of H₂S.

2. Purification of Pf DNA Polymerase I

A. Lysis of Pf Cell Paste

Fifty grams (g) of Pf cell paste prepared in Example 1 were thawed atroom temperature. Two hundred milliliters (ml) of lysis bufferconsisting of 50 millimolar (mM) Tris-HCl, pH 8.2, 10 mM betamercaptoethanol, 1 mM EDTA and 200 microgram/ml (μg/ml) of lysozyme wereadmixed to the thawed cell paste. The admixture was thereaftermaintained for 30 minutes at room temperature. The maintained admixturewas processed in a French press for two cycles. The cell lysate wassonicated for 10 minutes at room temperature and centrifuged at 16,000RPM in a SA600 rotor for 60 minutes at room temperature and thesupernatant recovered.

B. Column Chromatography of Pf Cell Lysate

The supernatant prepared above was loaded on to a Q-sepharose (2.5×40centimeter) column at room temperature. The column containing the celllysate supernatant was then washed with 200 ml of column buffer (50 mMTris-HCl, pH 8.2, 10 mM beta mercaptoethanol and 1 mM EDTA). The columnpass through and the washes were collected, pooled, and then centrifugedat 9000×g in a Sorvall GS3 rotor at room temperature to remove anyinsoluble material.

The resulting supernatant, containing partially purified Pyropolymerase, was recovered from the pellet and loaded directly onto aphosphocellulose column (2.5×40 cm) at room temperature. The column waswashed with column buffer to remove any proteins that did not bind tothe column until the optical density measured at an absorbance of 280 nmdropped to baseline. The immobilized Pyro polymerase was thereaftereluted with a one liter linear gradient of Nacl ranging in concentrationfrom 0 M to 0.7 M dissolved in column buffer and 10 ml fractions werecollected.

C. Assay for Pfu DNA Polymerase I Activity

The collected fractions were separately assayed for Pyro polymeraseactivity. The following reagents were admixed to form a reactioncocktail for the measurement of Pyro polymerase activity; finalconcentrations (fc) of the reagents in the cocktail are in parentheses:

(1) 200 microliters (μl) active calf thymus DNA, 575 μl distilled water,20 μl 1 M Tris-HCl, pH 7.5 (fc=20 mM); 8 μl 1 M MgCl₂ (fc=8 mM);

(2) 10 μl 0.75 M DTT (fc=7.5 mM); 4 μl 15 mg/ml BSA (fc=50 μg/ml);

(3) 30 μl 10 mM each of dATP, dCTP, dGTP dTTP (fc=0.15 mM for each); and

(4) 50 μl ³H-TTP in ethanol 432A (Amersham Inc., Arlington Heights,Ill.) for a total volume of 1 ml.

To perform the Pyro polymerase (DNA Polymerase I) activity assay, 25 μlof the reaction cocktail formed above was admixed with 1 to 5 ul of eachcollected fraction. The admixture was maintained for 10 to 60 minutes at75° C. which was the optimal temperature for enzymatic activity to forma labelled DNA admixture. After the maintenance period, 2 to 5 μl of thelabeled DNA admixture was pipetted onto DE81 (Whattman) filter paper.The filter paper containing the labeled DNA admixture was dried onaluminum foil under a heat lamp for 5 minutes. After the drying period,each filter was washed three times for 5 minutes with 50 ml of 2×SSC(0.3 M NaCl, 0.03 M NaCitrate) followed by one quick wash with 100% coldethanol. The washed filters were immediately placed fresh aluminum foiland placed under a heat lamp for 5 minutes to dry the filters. The driedfilters were separately placed in scintillation vials containing 5 mlscintillation fluid.

The ³H-DNA immobilized on the filter paper, which reflects Pyropolymerase activity, was measured in a scintillation counter. Theresults of this assay indicated that the peak fractions from thephosphocellulose column containing the highest concentration of Pyropolymerase were eluted with 200 mM NaCl and that Pyro polymeraseconstituted about 10% of the total protein present in those fractions.

D. FPLC Purification of Pyro Polymerase

The fractions containing approximately 90% of the total DNA polymerase Iactivity as measured above were pooled and dialyzed against columnbuffer overnight at 4° C. to form a NaCl-free Pyro polymerase solution.The dialyzed salt-free Pyro polymerase solution was loaded onto a Mono SHR 5/5 FPLC (fast phase liquid chromatography) column (Pharmacia,Piscataway, N.J.) previously equilibrated with the before-describedcolumn buffer. The Mono S column containing the Pyro polymerase waswashed with about four column volumes of column buffer prior to elutionto remove any proteins that did not bind to the column. The immobilizedproteins were eluted with a one liter linear gradient of NaCl ranging inconcentration from 0.0 M to 0.7 M dissolved in column buffer.

Fractions were collected and assayed for the presence of FPLC purifiedPyro polymerase activity as described, respectively, in B and C above.The results of this assay indicated that the peak fractions from theMono S column containing the highest concentration of FPLC purified Pyropolymerase were eluted with 120 mM NaCl. The fractions containing 90% ofthe peak FPLC purified Pyro polymerase activity were pooled and dialyzedagainst the column buffer additionally containing 10% glycerol overnightat room temperature to form NaCl-free FPLC purified Pyro polymerase.

The resultant purified and dialyzed Pyro polymerase was then subjectedto a final purification on a 1.5×20 cm Matrix gel Blue A column (Amicon,Danvers, Mass.). The Matrix gel Blue A column was first equilibratedwith the before-described column buffer containing 10% glycerol, 0.1%Tween 20 (polyoxyethylenesorbitan monolaurate) and 0.1% non-idet P40(octylohenol-ethyl ene oxide condensate containing an average of 9 molesethylene oxide per mole of phenol). The purified and dialyzed Pyropolymerase was then applied to the column using FPLC pumps. The columncontaining the Pyro polymerase sample was then washed with two columnvolumes of the glycerol-containing column buffer to remove any proteinsthat did not bind to the column. The immobilized Pyro polymerase waseluted from the column with a one liter linear gradient of KCl rangingin concentration from 0.0 M to 0.7 M KCl.

Eluted fractions from the Affi-gel column were collected and assayed forthe presence of purified Pyro polymerase activity as described inExamples 2B and C above. The fractions eluted with 200 to 300 mM KClcontained the peak Pyro polymerase activity, with the optimal activityrecovered at about 280 mM KCl. The peak fractions were pooled andconcentrated through Centricon-30 columns which have a molecular weightcut-off at 30,000 kD (Amicon, Beverly, Mass.) to form a concentratedsolution of purified Pyro polymerase. The purified Pyro polymerase wasthereafter dialyzed against column buffer containing 50% glycerol toform KCl-free purified Pyro polymerase. The resultant salt-free Pyropolymerase was determined to be about 95% homogeneous.

3. Molecular Weight Determination

The molecular weight of the purified Pyro polymerase prepared in Example2D was determined by SDS-PAGE under non-denaturing conditions accordingto the method of Laemmli et al., J. Mol. Biol., (1973) 80:575–599.Samples of Pyro polymerase, Taq polymerase, phosphorylase B, and bovineserum albumin, were applied to a 6–18% gradient, 1 mm thick,SDS-polyacrylamide gel (Novex, Encinitas, Calif.) and electrophoresed ina running buffering containing 1% SDS, 2.4 mM Tris, and 18 mM Glycine.The results of that analysis, shown in FIG. 1, indicate that Pyropolymerase migrates faster than phosphorylase B (Sigma, St. Louis Mo.;molecular weight 97,200 daltons) and Taq polymerase (Perkin-Elmer Cetus,Norwalk, Conn.; 94,000 daltons), but slower than BSA (Sigma, 66,000daltons). Because of its proximity to Taq, Pyro polymerase was assigneda relative molecular weight of 90,000–93,000 daltons.

4. Fidelity Assays

Various assays were performed to complete the characterization of DNAPolymerase I purified from P. furiosus. In the assays described below,the Pyro polymerase was compared to the commercially available and wellcharacterized Thermus aquaticus (Taq) DNA polymerase (U.S. Pat. No.4,889,818).

To determine the error rate of Pyro polymerase, fidelity assays by PCRwere performed with the Pyro polymerase in an assay procedure generallydescribed by Kohler et al, Proc. Natl. Acad. Sci. USA. 88:7958–7962(1991), in which in vivo mutagenesis was monitored during PCRamplification of transgenic mouse genomic DNA containing 33 copies percell of the lacIOZα transgene. The entire lac I gene plus thealpha-complementing fragment of the beta-galactosidase gene wasamplified (30 to 40 rounds) by PCR to form amplified DNA. The amplified1.9 kb DNA was then cloned into the EcoR I site of lambda gt10 andplated on host strain DH5alpha (lacZ▴M15) which contains the alphafragment of beta-galactosidase.

The complementation of the two proteins resulted in enzymically-activebeta-galactosidase, which was detected as blue plaques when X-gal(5-bromo-4-chloro-3-indolyl-beta-D-galactosidase) was present as asubstrate. In contrast, a functional non-mutant lac I repressed theexpression of beta-galactosidase which resulted in white plaques. Themutation frequency in lac I was defined as the proportion of mutant blueplaques to the total number of plaques scored. The error rate was thencalculated by the formula E=2 (mf/d) where mf was the observed mutationfrequency in the PCR product (lac I) and d was the number of doublingsaccording to Saiki et al., Science, 239:487–491 (1988).

Results from these studies indicate that Pyro polymerase has an errorrate (mutations per nucleotide per PCR cycle) as low as 1×10⁻⁶ comparedto 1×10⁻⁵ for Taq DNA polymerase as described by Eckert et al., Nucl.Acids. Res., 18:3739–3744 (1990). Thus Pyro polymerase exhibits about aten-fold greater replication fidelity than Taq DNA polymerase.

5. Exonuclease 3′ to 5′ Activity Assays

Purified Pyro polymerase, prepared as in Example 2D, was assayed tomeasure its 3′ to 5′ exonuclease activity. The exonuclease assay wasconducted according to Chase et al, J. Biol. Chem., 249:4545–4552(1974), except that the reaction was at 72° C. instead of 37° C. and thesubstrate was Taq I-digested lambda DAN filled in with tritium labelleddCTP and dGTP. Briefly, the following reagents were admixed to form areaction cocktail for the measurement of 3′ to 5′ exonuclease activity;final concentrations (fc) of the reagents in the cocktail are inparentheses:

-   -   (1) 40 μl 1 M Tris-HCl, pH 7.5 (fc=40 mM);    -   (2) 10 μl 1 M MgCl₂ (fc=10 mM);    -   (3) 13.3 μl 0.75 M DTT (fc=10 mM);    -   (4) 100 μl labelled Taq 1 cut lambda DNA filled in with        Sequenase™ and labelled with ³H dCTP and ³HdGTP obtained from        Amersham, Arlington Heights, Ill.; and    -   (5) 636.7 μl distilled water for a total volume of 1 ml.

For the 3′ to 5′ exonuclease activity assay, 20 to 25 μl of the preparedreaction cocktail was admixed with either Pyro or Taq DNA polymerase.The admixture was maintained for 10 to 60 minutes at 72° C. to formhydrolyzed lambda DNA, specifically ³H-5′ phosphate mononucleotides. Thereaction in each admixture was terminated by admixing 5 μl of 15 mg/mlbovine serum albumin (BSA) and 13 μl of 50% trichloroacetic acid (TCA)and maintaining the admixture on ice for 15 minutes. The terminatedreaction admixture was then centrifuged at 12,000×g for 15 minutes topellet the unhydrolyzed intact lambda DNA.

The resultant supernatant containing the ³H-5′exonuclease-derivedphosphate mononucleotides was removed from the pellet. Forty μl of eachsupernatant was admixed with 80 ul distilled water and 1 mlscintillation fluid. The amount of ³H radioactivity detected byscintillation counting was a relative measure of the exonucleaseactivity of the Pyro and Taq DNA polymerase preparations.

The results of this study show that Pyro polymerase exhibits detectable3′ to 5′ exonuclease activity, whereas Taq polymerase does not.

To determine the amount of non-specific nuclease activity present in thePyro polymerase preparation, Pyro polymerase prepared in Example 2D wasadmixed with 20 to 25 μl of a reaction cocktail. The following reagentswere admixed to form the reaction cocktail for the measurement ofnon-specific nuclease activity; final concentrations (fc) of thereagents in the cocktail are in parentheses:

-   -   (1) 40 μl 1 M Tris-Hcl, pH 7.5 (fc=40 mM);    -   (2) 10 μl 1 M MgCl₂ (fc=10 mM);    -   (3) 13.3 μl 0.75 M DTT (fc=10 mM);    -   (4) 100 μl ³H-labelled E. coli chromosomal DNA (sheared ten        times through a 21 gauge needle); and    -   (5) 835.7 μl distilled water for a final volume of 1 ml.

The admixture containing the reaction cocktail and the homogenized gelwere maintained for 10 to 60 minutes at 75° C. which was the optimaltemperature for the enzyme to form hydrolyzed nucleic acid product. Thereaction in the admixture was terminated and supernatant was assayed asdescribed in Example 2C above. The results of this assay show that Pyropolymerase did not exhibit detectable non-specific nuclease activity.The 3′ to 5′ exonuclease activity by Pyro polymerase is, therefore,specific and not due to non-specific nuclease activity.

Thus, pfu DNA polymerase I is a thermostable DNA polymerase that, unlikeTaq DNA polymerase, possesses a 3′ to 5′ exonucleases activity whichenables pfu polymerases to proofread errors, and thereby exhibits aten-fold greater fidelity during DNA synthesis reactions.

6. PCR with Pyro Polymerase

The specificity of Pyro polymerase was evaluated in PCR amplification oftemplate DNA compared to that achieved with Taq DNA polymerase. Toprepare the template DNA for the reactions, genomic DNA was firstpurified by phenol extraction and alcohol precipitation according to theprocedures of Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons (1987), from blood obtained by tail bleed from atransgenic mouse having about 1 to 2 copies of a lambda transgenevector, which is referred to as lambda transgene mouse genomic DNA.

The lz-lambda polynucleotide primers with the used in the PCRamplifications were prepared by chemical synthesis using a model 381Apolynucleotide synthesizer (Applied Biosystems Inc., Foster City,Calif.) according to the manufacturer's instructions.

A hybridization reaction admixture was formed by combining the followingreagents in a sterile 0.5 ml microfuge tube:

-   -   (1) 80 μl of sterile, autoclaved water;    -   (2) 10 μl of 10× reaction buffer containing 500 mM KCl, 100 mM        Tris-HCl, pH8.3, 15 mM MgCl₂, and 0.1% sterile gelatin;    -   (3) 8 μl of a solution containing 2.5 mM each of the        deoxynucleotidetriphosphates (dNTP's) dGTP, dCTP, dTTP, and        dATP;    -   (4) 1 μl of a solution containing 250 ng each of the two        polynucleotide primers described above; and    -   (5) 1 μl of lambda transgene mouse genomic DNA.

The hybridization reaction admixture was heated to 94° C. and maintainedat 94° C. for 1 minute to denature the duplex genomic DNA present andform single-stranded templates. The admixture was then cooled to 54° C.and maintained for 2 minutes to allow hybridization to occur and formduplex DNA. The hybridized admixture was thereafter centrifuged in amicrofuge at 12,000×g for 10 seconds to collect condensation off themicrofuge tube walls. To separate microfuge tubes, 0.5 ul of a solutioncontaining 2.5 units of either Taq DNA polymerase (obtained fromPerkin-Elmer Cetus, Norwalk, Conn. or from Stratagene, La Jolla, Calif.)or Pyro polymerase prepared in Example 2D was admixed to form a primerextension reaction admixture. Additional separate primer extensionreaction admixtures were made by diluting the amount of Pyro polymerasefrom 1:1.5, 1:2, 1:2.5, 1:3, 1:4 and 1:5 to determine the optimalconcentration of DNA synthesis. (The 1:3 dilution represented about 1.5unit of Pyro polymerase per microliter.)

Each microfuge tube containing the above-prepared primer extensionreaction admixture was overlayed with 50 μl of mineral oil and thenplace into a DNA Thermal Cycler (Perkin-Elmer Cetus) and subjected tothe following temperature and time conditions: 1) 94° C. for 1 minute todenature duplex DNA; 2) cooled to 54° C. for 2 minutes to anneal theprimers; and 3) heated to 74° C. for 1.0 minute to activate thepolymerase and maintained at 74° C. for 0.5 minutes to extend theannealed primers. The tubes were subjected to 30 cycles of the abovesequence according to the manufacturer's instructions. The cycled tubeswere then maintained at 72° C. for 10 minutes followed by 4° C. for 12hours.

The contents of each primer extension reaction admixture were analyzedon a 6% polyacrylamide gel in 1× TBE by loading 35 μl of the admixturesample and 5 μl of 10× sample buffer onto an 8 centimeter gel,electrophoresing the gel at 100V for about one hour followed by stainingthe electrophoresed gel with ethidium bromide to visualize theelectrophoresed nucleic acids.

The results of the electrophoresed PCR amplified lambda transgene mousegenomic DNA using either Pyro or Taq DNA polymerase indicted that theTaq from Cetus and Stratagene gave nearly identical results. The resultsusing the Pyro DNA polymerase to amplify genomic DNA indicate that itproduces less background. The 1:2.5 dilution of Pyro polymerase resultedin optimal PCR amplification.

7. Large Scale Preparation of Pyrococcus furiosus DNA Polymerase I

The following steps 1–11 are performed at room temperature:

1. Thaw 1500 grams of frozen cell paste at room temperature. Add 4volumes (6000 mls) of lysis buffer A.

2. Resuspend the cells and incubate at room temperature for 30 minutes.Cycle the cells through a French press two times.

3. Sonicate the product of step 2 for 10 minutes at room temperature.Centrifuge the resulting lysate at 9K RPMs in a GS3 (Sorvall) rotor atroom temperature for 60 minutes.

4. Collect the supernatant (Fraction I) and load it directly onto aQ-sepharose (8×30 cm) column. Wash the loaded column with 4 volumes (6liters) of buffer B.

5. Collect the pass-through and washes. Adjust the collected material topH 6.0 with HCl, and centrifuge it at 9K in a GS3 rotor at roomtemperature for 60 minutes to remove the protease-containingprecipitate.

6. Collect the supernatant and adjust its pH to 7.8 with NaOH (FractionII). Load it at 50 ml/minute directly onto a 500 ml radial flow P-11(phosphocellulose) (Sepragen, San Leandra, Calif.) column equilibratedwith buffer B. Wash the column with 1–2 liters of buffer B until theOD₂₈₀ of the wash is back to baseline.

7. Elute DNA polymerase activity with a 0–0.7M NaCl gradient in buffer B(2×2500 ml). The peak of polymerase activity elutes at approximately0.2–0.4M NaCl.

8. Assay and pool those fractions containing greater than 90% of thetotal polymerase activity.

All subsequent steps are performed at 4° C.

9. Dialyze the pooled material of step 8 overnight against 200 volumesbuffer B at 4° C.

10. Remove the dialyzed Pyro polymerase and clarify it with a 30 minutecentrifugation if necessary (Fraction III). Load dialysate onto aheparin-agarose column (5×20 cm) equilibrated with buffer B. Wash with 1liter buffer B and elute with a 0–0.7M NaCl gradient in buffer B (2×1.5liters).

11. Assay for polymerase activity, and pool the fractions containinggreater than 90% of total activity. Dialyze the recovered materialagainst 200 volumes of buffer C.

12. Remove the Pyro polymerase from dialysis (Fraction IV) and load itonto an Affigel Blue column (2.5×20 cm) equilibrated with buffer C.

13. Wash the Affigel-Blue column with buffer C until the OD 280approaches baseline and elute the Pyro polymerase with a 0–0.7M KClgradient in buffer C 2×1000 ml).

14. Assay for polymerase and 3′ exonuclease activities. Analyze theactive fractions by silver stained SDS-PAGE gels. Pool the pure Pyropolymerase (greater than 90% pure on a w/w basis) fractions based onvisual analysis silver stained gel.

15. Dialyze overnight against 200 volumes of buffer D at 4° C. Removethe dialysate (Fraction V) and load it onto an 1.5×30 cm P11(phosphocellulose) column equilibrated with buffer D. Wash with buffer Duntil wash OD 280 is baseline. Elute the Pfu DNA PolI with a 2×250 mllinear gradient 0.0˜0.7M kCl prepared in buffer D.

16. Assay for polymerase and 3′ exonuclease activities. Analyze theactive fractions by silver stained SDS-PAGE gels. Pool the pure Pyropolymerase (greater than 95% pure on a w/w basis) fractions based onvisual analysis silver stained gel.

17. Pool and dialyze overnight at 4° C. against 20 volumes buffer E.Remove from dialysis (fraction VI) and store at −20° C.

BUFFERS Buffer A: (6 liters) 50 mM Tris-Cl pH 8.2 10 mM betamercaptoethanol 1 mM EDTA 200 μg/ml lysozyme Buffer B: (20 liters) 50 mMTris-Cl pH 8.2 10 mM beta mercaptoethanol 1 mM EDTA Buffer C: (6 liters)50 mM Tris-Cl pH 8.2 10% glycerol 1 mM EDTA 1 mM DTT 0.1% tween 20 0.1%nonidet P40 Buffer D: (2 liters) 50 mM Tris-Cl pH 8.2 10% glycerol 1.0mM EDTA 10 mM beta mercaptoethanol 0.1% tween 20 0.1% nonidet P40 BufferE 50 Tris Hcl pH 8.2 0.1 mM EDTA 1 mM DTT 0.1% Tween 20 0.1% NP 40 50%glycerol RESINS 1.5 LITERS Q-SEPHAROSE (PHARMACIA) 700 MLS P-11(WHATMANN) 200 MLS HEPARIN-AGAROSE (BIORAD) 200 ML AFFIGEL-BLUE (BIORAD)8. Production of Pfu I

Cell Growth: Pyrococcus furiosus (DSM 3638) was grown at 85–88° C. asclosed static cultures in a medium containing maltose (5 g/liter), NH₄Cl(1.25 g/liter) elemental sulfur (S^(o), 5 g/liter), Na₂S (0.5 g/liter),synthetic sea water (17), a vitamin mixture (18), FeCl₃ (25 mM), NaWO₄(10 mM) and yeast extract (1.0 g/liter). Growth was monitored by directcell count and by the increase in turbidity at 600 nm. For large scalecultures, sulfide was replaced with titanium (III) citrate, and S^(o)was omitted which necessitated sparging with Ar. Two 20 liter culturesserved as an innoculum for growth in a 400 liter fermenter where thecultures were maintained at 88° C., bubbled with Ar (7.5 liters/min) andstirred (50 rpm). Cells were harvested after approximately 20 hours(OD₆₀₀-0.5) with a Sharples centrifuge at 100 liters/hour.

NOTE: The following procedures are performed at 25° C., unless otherwisestated.

Cell Lysis: The night before lysis, 500 grams of frozen cell paste istransferred to a 2–8 C refrigerator. The next morning, the cells aretransferred to a 4 liter stainless steel beaker. The cells areresuspended using 4 volumes (2000 ml) of lysis Buffer 8A. The cellsuspension is then incubated at room temperature for 1.5 hours. Thecells are lysed in the French Press using 2 passes at 8K PSI. The lysateis then sonicated at room temperature for 10 minutes.

Following sonication, the lysate is transferred to 400 ml bottles andspun for 60 minutes at 9K rpm at room temperature in the Sorvall RC-2Busing a Sorvall GS3 rotor. The supernatant (Fraction I) is collected andthe volume measured.

Q-Sepharose Column: Fraction I is loaded directly onto a 8×30 cm radialflow Q-sepharose column (_(—)1500 ml) pre-equilibrated in Buffer 1B at aflow rate of approximately 50 ml/minute. The column is then washed with4 column volumes (6000 ml) of Buffer 8B.

NOTE: It is important to carefully collect the pass-thru and washfractions as they contain the Pfu polymerase enzyme

The Q-Sepharose pass-thru and column washes are next combined as onepool (Fraction II).

BPA-1000 Precipitation: Bioprocessing aid BPA-1000 (TosoHaas) pilots areconducted on Fraction II to determine the appropriate volume required toprecipitate cell debri and nucleic acids but not Pfu polymerase. 1.0 mlaliquots of Fraction II are placed in 8 tubes. The BPA-1000 is mixedthoroughly and 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 ml of BPA-1000are added to the tubes respectively, mixed gently, incubated for 5minutes, and spun in a microfuge for 5 minutes. Carefully decant andsave the supernatants. Samples of each tube are then evaluated using thegapped duplex polymerase assay. Evaluate the clarity of each supernatantand the firmness of each pellet. Based on the control tube (without BPAaddition), determine which concentration of BPA increases the clarity ofthe supernatant without sacrificing polymerase yield. The appropriateamount of BPA-1000 is then added to Fraction II and stirred for 30minutes. The suspension is then transferred to 400 ml centrifuge bottlesand spun at 9K rpm at room temperature (25° C.) for 60 minutes in aSorvall RC-2B using a Sorvall GS3 rotor. The supernatant (Fraction III)is collected and the volume measured.

Fraction III is then adjusted to pH 7.5 with 1 N HCl if necessary.

P-11 Cellulose Column: Fraction III is loaded directly onto a 10×14 cmP-11 column (⁻1000 ml) pre-equilibrated in Buffer 8C at a flow rate ofapproximately 5 ml/minute. The column is washed with Buffer 8C until theOD₂₈₀ approaches baseline (4 column volumes). The column is next elutedwith a 2×4000 ml gradient (0 to 700 mM KCl) prepared in Buffer 8C. 25 mlfractions are collected, every fifth tube, put-on and pass-thru areassayed for polymerase activity. The fractions containing the peak ofPfu Pol I activity are pooled and concentrated to approximately 200 mlusing an Amicon model CH₂ concentrator with a SIY30 membrane cartridge.Any remaining Pfu Pol I is washed from the concentrator by flushing thelines with an additional 300 ml Buffer 8C. The concentrate and wash arecombined (Fraction IV).

NOTE: The following procedures are performed at 4° C.

Fraction IV is next dialyzed overnight against 18 liters of Buffer D.

Hi-Load S Column: The following morning the dialysate is transferred to400 ml centrifuge bottles and spun 9K rpm at 4° C. for 60 minutes in aSorvall RC-2B using the GS3 rotor. The supernatant (Fraction V) isrecovered and the volume recorded. Fraction V is divided into two equalportions. The first portion is loaded directly onto a FPLC Hi-Load Scolumn (⁻58 ml) pre-equilibrated in Buffer D at a flow rate of 5.0ml/minute. The column is washed with Buffer 8D until the OD₂₈₀approaches baseline. The column is next eluted with a 2×250 ml gradient(0 to 250 mM KCl) prepared in Buffer D. 5.0 ml fractions are collected,every third tube, put-on and pass-thru are assayed for polymeraseactivity. The above procedure is repeated for the second portion ofFraction V. The fractions containing the peak Pfu Pol I activity arepooled and dialyzed overnight against 2×4 liters of Buffer 8E at 4 C.The following morning, the dialysate is removed from dialysis and thevolume recorded (Fraction VI).

Heparin Sepharose CL-6B Column: Fraction VI is loaded onto a 1.5×90 cmheparin sepharose CL-6B column (⁻159 ml) pre-equilibrated in Buffer 8Eat a flow rate of 0.5 ml/minute. The column is washed with 1000 mlBuffer 8E. The column is next eluted with a 2×750 ml gradient (0 to 300mM KCl) prepared in Buffer 8E. 10 ml fractions are collected, everythird tube, put-on and pass-thru are assayed for polymerase activity. Aprotein gel is also recommended for peak evaluation. The fractionscontaining the peak Pfu DNA polymerase activity are pooled and dialyzedovernight against 2×4 liters of Buffer 8E. The following morning, thedialysate is removed from dialysis and the volume recorded (FractionVII).

Affi-Gel Blue Column: Fraction VII is loaded onto a 2.5×4.0 cm affi-gelblue column (⁻20 ml) pre-equilibrated in Buffer 8E at a flow rate of 0.5ml/minute. The column is washed with Buffer 8E until the OD₂₈₀approaches baseline. The column is next eluted with a 2×500 ml lineargradient (0 to 250 mM KCl) prepared in Buffer 8E. 10 ml fractions arecollected, every third tube, put-on and pass-thru are assayed forpolymerase activity. A SDS-PAGE gel is also recommended for careful peakevaulation. The fractions containing the peak Pfu Pol I activity arepooled and dialyzed overnight against 1 liter final dialysis Buffer SF.The following morning, the purified enzyme is removed from dialysis andtransferred to −20° C. storage and represents a purified pfu polymeraseI enzyme of this invention.

Buffer 8A: Lysis Buffer 50 mM Tris-Cl, pH 8.2 1 mM EDTA 10 mMb-mercaptoethanol 200 mg/ml lysozyme Buffer 8B: Q-Sepharose Buffer 50 mMTris-Cl, pH 8.2 1 mM EDTA 10 mM b-mercaptoethanol Buffer 8C:Phosphocellulose Buffer 50 mM Tris-Cl, pH 7.5 1 mM EDTA 10 mMb-mercaptoethanol Buffer 8D: High Load S Buffer 50 mM Tris-Cl, pH 7.5 1mM EDTA 1 mM DTT 10% (v/v) glycerol 0.1% (v/v) NP-40 0.1% (v/v) Tween 20Buffer 8E: Affigel Blue & Heparin Buffer 50 mM Tris-Cl, pH 8.2 1 mM EDTA1 mM DTT 10% (v/v) glycerol 0.1% (v/v) NP-40 0.1% (v/v) Tween 20 Buffer8F: Final Dialysis Buffer 50 mM Tris-Cl, pH 8.2 0.1 mM EDTA 1 mM DTT0.1% (v/v) NP-40 0.1% (v/v) Tween 20 50% (v/v) glycerol9. Cloning the Gene that Encodes Pyrococcus furiosus (Pfu) DNAPolymerase I

Pyrococcus furiosus (DSM 3638) was grown as described by Bryant et al,J. Biol. Chem., 264:5070–5079 (1989), with an additional supplement of10 mM Na₂WO₄ as described in the Examples. Following harvesting bycentrifugation, genomic DNA was isolated from the biomass usingStratagene's genomic DNA isolation kit according to manufacturer'instructions. The DNA was then randomly sheared by several passagesthrough an eighteen gauge needle and the fragments were separated bysucrose gradient centrifugation. The size of the fragments presentwithin the fractions of the sucrose gradient were next estimated byagarose gel electrophoresis. The fractions containing four to ninekilobase fragments were combined and ligated to EcoR1 linkers and theresulting inserts were ligated into EcoR1 cut Lambda Zap II vector(Stratagene, La Jolla, Calif.) to create a genomic Pyrococcus furiosuslibrary. This library was plated with XL1-Blue E. coli (Stratagene) onLB plates. Plaque lifts were performed on Duralose nylon filters(Stratagene) to isolate individual bacteriophage colonies containing acloned insert.

N-terminal amino acid sequence determination of pfu I purified asdescribed in Example 2 was performed by the Wistar Institute. Briefly,partially purified protein was subjected to SDS-PAGE followed byelectrotransfer to a nylon membrane. The band corresponding to Pfupolymerase was isolated and subjected to protein microsequencing. Fromthis microsequencing analysis, the unambiguous sequence of theforty-eight N-terminal amino acids of the pfu polymerase I protein wasdetermined. The 48 residues corresponds to residues 1 to 48 of SEQ IDNO 1. Internal amino acid sequence analysis was also performed by theWistar Institute on tryptic digested fragment of the Pfu polymeraseprotein.

Based on the N-terminal 48 amino acid residue sequence information, aseries of degenerate PCR oligonucleotide primers were designed in pairswhich would produce a 94 basepair (bp) PCR product. The 94 bp productcorresponds to amino acid residues within the 48 residues. Theseoligonucleotides (23 and 18 bases corresponding to the two ends of the94-mer) were designed such that the 3′ terminal 8 nucleotides of everysequence possible based on the known amino acid residue sequence withinthe 48 residues was present as a separate oligonucleotide. The 23-mercorresponds to possible nucleotides at positions 224 to 246, and the18-mer corresponds to possible nucleotides at positions 300 to 317.Whenever there was a wobble position in the rest of the oligonucleotideprimer, a T was used. The rationale for these substitutions was based onthe fact that the GC content of pfu is considerably low and that Tmismatches are most tolerated by Taq polymerase. In this way, fouroligos were needed for each of the two primer positions. Each of theseoligos contained some mismatched bases but no degenerate positions.

The PCR was performed on pfu genomic DNA with all 16 possible primerpairs. Following agarose gel electrophoresis, it was determined that 12of the 16 amplification reactions produced the expected 94 bp PCRproduct. One of the reactions containing the 94 bp product was subjectedto direct cycle sequencing (Stratagene) using both PCR primers used inthe amplification reaction as sequencing primers. A 53 base sequencededuced from the DNA sequencing gel was found to corresponded 100% tothe known N-terminal amino acid sequence between a pair of primers, thusconfirming that the sequence was from the gene encoding Pfu DNApolymerase. A 53 base oligonucleotide probe containing this sequence wasnext synthesized, and having the nucleotide sequence shown in SEQ ID NO2 from nucleotides 247 to 299. This oligonucleotide was then endlabelled with ³²P and used to screen the Pfu genomic library.Approximately four Pyrococcus genomes were represented in the screenedlibrary. The probe was hybridized to plaque lifts at 42° C. for twohours in QuikHyb (Stratagene) and washed twice at room temperature for15 minutes each in 2×SSC 0.1% SDS. Thirteen putative clones wereidentified and the plaques cored and resuspended in 300 ml SM. Ten ml ofeach lysate was transferred into 1× Taq reaction buffer, heated to 100°C. for ten minutes, and 30 cycles of PCR was performed using the PCRprimer pair which originally produced the expected 94 bp fragment (PCRwas performed according to the procedure described in the GeneAmp kit[Cetus Perkin-Elmer]). Following electrophoretic analysis of the PCRproducts, three clones produced the expected 94 bp PCR product. Thesethree lambda clones were excised into to a pBluescript plasmid(Stratagene) according to the manufacturer's protocol. The resultingtransformants were then screened by PCR. PCR was performed on singlecolony transformants (100° C. in 1× Taq reaction buffer for 10 minutes,then centrifuged briefly followed by 30 cycles of PCR using the originalprimer pair and the procedure described in the GeneAmp kit. Threepositive plasmid clones were identified from each excision and largescale plasmid preparations were performed on one colony from each clone.

The three bonafide polymerase clones were next mapped. One had arelatively small 1500 bp insert. The other two clone inserts were about4500 basepairs; one containing about 1000 bp of the Pfu polymerasesequence while the other clone contained the entire polymerase gene. Theclone having the entire pfu polymerase I gene was named pF72.

The insert of clone pF72 was sequenced on both strands using Sequenase™(USB) and custom oligonucleotide primers using a primer walkingstrategy. The sequence of the polymerase gene is shown in SEQ ID NO 2from nucleotide bases 224 to 2548, and consists of a 2265 bp DNA seqmentencoding 775 amino acids corresponding to a 90,113 Dalton protein.

The Pfu gene can be cloned into an expression system for production ofthe recombinant protein. The complete coding region of the pfupolymerase I gene is PCR-amplified using Pfu polymerase to limitmutations and the PCR product product is ligated in reading frame intothe vector pRSET (Invitrogen). The ligated vector is transformed into anF′ containing E. coli host strain and protein production is induced witha recombinant M13 phage expressing cloned T7 RNA polymerase undercontrol of the lacUV5 promoter. The expression vector is designed tointroduce an affinity tail which can be used to facilitate purificationof the recombinant protein. Following cell lysis and clarification ofthe crude supernatant, the recombinant Pfu polymerase protein isisolated in a single step by metal affinity chromatography.

In another strategy, the coding region of the Pfu DNA polymerase gene isamplified with Pfu polymerase using primers which introduce a uniquerestriction site at the ends of the PCR product to facilitate cloninginto a pBluescript vector. When cloned into the T3 orientation, theprotein is expressed under control of the lacZ promoter. Followingexpression, recombinant Pfu polymerase is purified with a modificationof the procedure used to purify the native enzyme. Briefly, a heatprecipitation step is employed following cell lysis and clarification.The heat precipitation step denatures and precipitates the majority ofE. coli host proteins but not the thermostable Pfu polymerase and theremaining soluble fraction is recovered and used as described before asa source for purification of the pfu polymerase I protein.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications can be effectedwithout departing from the true spirit and scope of the invention.

1. An isolated polynucleotide encoding a thermostable DNA polymerase,wherein the isolated polynucleotide is capable of hybridizing to apolynucleotide complementary to a polynucleotide that encodes aminoacids 1 to 775 of SEQ ID NO: 1 under hybridization conditions comprisingincubating at 42° C. in a hybridization buffer comprising 0.9 M NaCl and50% formamide, and under wash conditions comprising incubating at 68° C.in a wash buffer comprising 0.015 M NaCl and 0.5% sodium dodecylsulfate.
 2. The isolated polynucleotide of claim 1, wherein thethermostable DNA polymerase is a fusion protein.
 3. A vector comprisingthe isolated polynucleotide of claim
 1. 4. A host cell comprising theisolated polynucleotide of claim
 1. 5. A method of preparing arecombinant thermostable DNA polymerase, comprising culturing the hostcell of claim 4 under conditions suitable for the expression of thethermostable DNA polymerase.